2012 Volume 29 Issue 1 Pages 35-42
In tobacco (Nicotiana tabacum), several transcription factors belonging to the IXa group of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) cluster at the regulatory NIC2 locus for the leaf nicotine level, and directly activate structural genes required for nicotine biosynthesis and transport. Solanaceous Atropa and Hyoscyamus plants synthesize medicinal tropane alkaloids by utilizing enzymes that partially overlap with the nicotine pathway. We tested whether the tobacco NIC2-locus ERF189 binds to potential ERF binding sites in the promoters of various structural genes involved in biosynthesis of nicotine and tropane alkaloids by an electrophoresis mobility shift assay. ERF189 bound four new GCC-box-like sequences in three nicotine structural genes, but did not recognize other candidate binding sites. This binding preference was used to optimize the consensus binding sequence of ERF189 as 5′-(A/C)GC(A/C)NNCC(A/T)-3′. Five group IXa ERFs (tobacco ERF189, tobacco ERF163, Catharanthus ORCA3, Arabidopsis AtERF13, and Arabidopsis AtERF1) were examined whether they bind to the ERF189-recognition site in the promoter of the tobacco putrescine N-methyltransferase gene in vitro, and transactivate the promoter in a transient expression assay using cultured tobacco cells. The more the protein sequences were similar to ERF189, the more effective these ERFs were in these functional assays. The transient expression assay also identified transactivation domains in an N-terminal acidic region of ERF189, and in a C-terminal Ser-rich region of AtERF13.